RESUMEN
BACKGROUND: Human and mouse natural killer (NK) cells have been shown to develop memory-like function after short-term exposure to the cocktail of IL-12/15/18 or to overnight co-culture with some tumor cell lines. The resulting cells retain enhanced lytic ability for up to 7 days as well as after cryopreservation, and memory-like NK cells (mlNK) have been shown to induce complete remissions in patients with hematological malignancies. No single phenotype has been described for mlNK and the physiological changes induced by the short-term cytokine or tumor-priming which are responsible for these enhanced functions have not been fully characterized. Here, we have generated mlNK by cytokine and tumor-priming to find commonalities to better define the nature of NK cell "memory" in vitro and, for the first time, in vivo. METHODS: We initiated mlNK in vitro from healthy donors with cytokines (initiated cytokine-induced memory-like (iCIML)-NK) and by tumor priming (TpNK) overnight and compared them by high-dimensional flow cytometry, proteomic and metabolomic profiling. As a potential mechanism of enhanced cytolytic function, we analyzed the avidity of binding of the mlNK to NK-resistant tumors (z-Movi). We generated TpNK from healthy donors and from cancer patients to determine whether mlNK generated by interaction with a single tumor type could enhance lytic activity. Finally, we used a replication-incompetent tumor cell line (INKmune) to treat patients with myeloid leukaemias to potentiate NK cell function in vivo. RESULTS: Tumor-primed mlNK from healthy donors and patients with cancer showed increased cytotoxicity against multiple tumor cell lines in vitro, analogous to iCIML-NK cells. Multidimensional cytometry identified distinct memory-like profiles of subsets of cells with memory-like characteristics; upregulation of CD57, CD69, CD25 and ICAM1. Proteomic profiling identified 41 proteins restricted to mlNK cells and we identified candidate molecules for the basis of NK memory which can explain how mlNK overcome inhibition by resistant tumors. Finally, of five patients with myelodysplastic syndrome or refractory acute myeloid leukemia treated with INKmune, three responded to treatment with measurable increases in NK lytic function and systemic cytokines. CONCLUSIONS: NK cell "memory" is a physiological state associated with resistance to MHC-mediated inhibition, increased metabolic function, mitochondrial fitness and avidity to NK-resistant target cells.
Asunto(s)
Memoria Inmunológica , Células Asesinas Naturales , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Neoplasias/inmunología , Neoplasias/terapia , Proteómica/métodos , Citocinas/metabolismo , Línea Celular Tumoral , Inmunoterapia/métodos , Fenotipo , RatonesRESUMEN
BACKGROUND: Tissue-specificity for fimbrial fallopian tube ovarian carcinogenesis remains largely unknown in BRCA1 mutation carriers. We aimed to assess the cell autonomous and cell-nonautonomous implications of a germline BRCA1 mutation in the context of cancer immunosurveillance of CD3- CD56+ natural killer (NK) cells. METHODS: Premenopausal BRCA1 mutation carriers versus age-matched non-carriers were compared. Daily urinary 5ß-pregnanediol levels were used to determine progesterone metabolomics across an ovarian cycle. Using peripherally acquired NK cells the cell-mediated cytotoxicity of tumor targets (OVCAR-3, K-562) was determined using live cellular impedance (xCELLigence®) and multicolor flow cytometry. Hypoxia-inducible factor 1-alpha (HIF-1α) immunohistochemistry of cancer-free fallopian tube specimens allowed a comparison of proximal versus distal portions. Utilizing these findings the role of environmental factors relevant to the fimbrial fallopian tube (progesterone, hypoxia) on NK cell functional activity were studied in an ovarian phase-specific manner. RESULTS: BRCA1 mutation carriers demonstrate a differential progesterone metabolome with a phase-specific reduction of peripheral NK cell functional activity. Progesterone exposure further impairs NK cell-mediated cytotoxicity in a dose-dependent manner, which is reversed with the addition of mifepristone (1.25 µM). The fimbrial fallopian tube demonstrated significantly higher HIF-1α staining, particularly in BRCA1 mutation carriers, reflecting a site-specific 'hypoxic niche'. Exposure to hypoxic conditions (1% O2) can further impair tumor cytotoxicity in high-risk carriers. CONCLUSIONS: Phase-specific differential NK cell activity in BRCA1 mutation carriers, either systemically or locally, may favor site-specific pre-invasive carcinogenesis. These cumulative effects across a reproductive lifecycle in high-risk carriers can have a detrimental effect further supporting epidemiological evidence for ovulation inhibition.
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An emerging cellular immunotherapy for cancer is based on the cytolytic activity of natural killer (NK) cells against a wide range of tumors. Although in vitro activation, or "priming," of NK cells by exposure to pro-inflammatory cytokines, such as interleukin (IL)-2, has been extensively studied, the biological consequences of NK cell activation in response to target cell interactions have not been thoroughly characterized. We investigated the consequences of co-incubation with K562, CTV-1, Daudi RPMI-8226, and MCF-7 tumor cell lines on the phenotype, cytokine expression profile, and transcriptome of human NK cells. We observe the downregulation of several activation receptors including CD16, CD62L, C-X-C chemokine receptor (CXCR)-4, natural killer group 2 member D (NKG2D), DNAX accessory molecule (DNAM)-1, and NKp46 following tumor-priming. Although this NK cell phenotype is typically associated with NK cell dysfunction in cancer, we reveal the upregulation of NK cell activation markers, such as CD69 and CD25; secretion of pro-inflammatory cytokines, including macrophage inflammatory proteins (MIP-1) α /ß and IL-1ß/6/8; and overexpression of numerous genes associated with enhanced NK cell cytotoxicity and immunomodulatory functions, such as FAS, TNFSF10, MAPK11, TNF, and IFNG. Thus, it appears that tumor-mediated ligation of receptors on NK cells may induce a primed state which may or may not lead to full triggering of the lytic or cytokine secreting machinery. Key signaling molecules exclusively affected by tumor-priming include MAP2K3, MARCKSL1, STAT5A, and TNFAIP3, which are specifically associated with NK cell cytotoxicity against tumor targets. Collectively, these findings help define the phenotypic and transcriptional signature of NK cells following their encounters with tumor cells, independent of cytokine stimulation, and provide insight into tumor-specific NK cell responses to inform the transition toward harnessing the therapeutic potential of NK cells in cancer.
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Células Asesinas Naturales/inmunología , Neoplasias/inmunología , Línea Celular Tumoral , Citocinas/genética , Citocinas/inmunología , Citotoxicidad Inmunológica , Redes Reguladoras de Genes , Humanos , Inmunoterapia , Mediadores de Inflamación/metabolismo , Células K562 , Células Asesinas Naturales/metabolismo , Activación de Linfocitos , Células MCF-7 , Neoplasias/genética , Neoplasias/terapia , Fenotipo , TranscriptomaRESUMEN
Two series of 2-thioxoimidazo[4,5-b]pyridine derivatives have been synthesized from 2,3-diaminopyridine (1) and 5-halogenosubstituted-2,3-diaminopyridines 2, 3. Mannich bases 7 - 12 and 24 - 29, derivatives of 1-arylamino-6-halogeno-2-thioxoimidazo[4,5-b]pyridine were obtained with selected secondary amines: morpholine, piperidine, 2-methoxyphenylpiperazine, pyrimidyn-2-yl-piperazine and formaldehyde in ethanol. The structures 7 - 12 and 24 - 29 were confirmed by the results of elementary analysis and their IR, 1H-NMR and MS spectra. All given structures 7 - 12 have been optimized to get the most stable low energy conformers. Synthesized compounds were of interest for biological studies or can be substrates for further synthesis. The selected compounds 7 - 10, 12- 17, 22, 25, 27 - 29 were screened for their antiproliferative activity in vitro against human cancer and normal mouse fibroblast cell lines.